Amino acid derivatives

ABSTRACT

New substituted acyl derivatives of amino acids which have the general formula ##STR1## are useful as angiotensin converting enzyme inhibitors. 
     SUMMARY OF THE INVENTION 
     This invention relates to new substituted acyl derivatives of amino acids which have the general formula ##STR2## and salts thereof, 
     WHEREIN R is hydroxy or lower alkoxy; 
     R 1  is hydrogen, lower alkanoyl or amino(imino)methyl; 
     R 2  is hydrogen, lower alkyl or phenyl-lower alkylene; 
     R 3  is hydrogen, lower alkanoyl, benzoyl or ##STR3## 
     A is hydrogen, lower alkyl or hydroxy-lower alkylene; 
     B is hydrogen, lower alkyl, phenyl, phenyl-lower alkylene, hydroxy-lower alkylene, hydroxyphenyl-lower alkylene, amino-lower alkylene, guanidino-lower alkylene, mercapto-lower alkylene, lower alkyl-thio-lower alkylene, imidazolyl-lower alkylene, indolyl-lower alkylene, carbamoyl-lower alkylene or carboxy-lower alkylene; 
     OR A and B together form a (CH 2 ) p  bridge which completes a ring of 5 or 6 atoms with the nitrogen and carbon to which they are joined, one carbon optionally bearing a hydroxy group; 
     N IS 0 OR 1; 
     M IS 0, 1, 2, 3 OR 4; AT LEAST ONE OF M AND N IS OTHER THAN 0; AND 
     P IS 3 OR 4. 
     The asterisks denote centers of asymmetry. 
     DETAILED DESCRIPTION OF THE INVENTION phenylglycine 
     The invention in its broad aspects includes substituted acyl derivatives of amino acids having formula I above. The substituted acyl groups refer to the side chains on the carbon beta to the nitrogen atom. The one side chain has one or two sulfur containing groups and the second side chain has one nitrogen containing group. Within the class defined by formula I, because of their properties, certain subgroups are preferred. 
     Compounds in the group represented by formula I which are derived from or include the structure of the amino acids glycine, alanine, leucine, threonine, phenylalanine, lysine, arginine, glutamine, histidine, methionine, serine, cysteine, tyrosine, valine, asparagine, glutamic acid, proline, hydroxyproline, pheylglycine or tryptophane are broadly preferred. Preferred modifications are compounds of formula I wherein R is hydroxy; R 1  is hydrogen, lower alkanoyl or amino(imino)-methyl [particularly hydrogen, methyl, acetyl or amino(imino)-methyl]; R 2  is hydrogen, lower alkyl (particularly hydrogen or methyl) or phenyl-lower alkylene (particularly benzyl), most particularly R 2  is hydrogen; R 3  is hydrogen, lower alkanoyl or benzoyl (particularly hydrogen, or acetyl); A is hydrogen; B is lower alkyl, guanidino-lower alkylene (particularly guanidinopropyl), amino-lower alkylene (particularly amino-C 3  -C 4  -lower alkylene) or phenyl-lower alkylene (particularly phenylmethyl); or A and B complete a 5- or 6-membered ring; m is 0, 3 or 4 and n is 0 or 1, but not both m and n are 0. 
     Especially preferred are those compounds of formula I which are derived from proline and have the formula ##STR4## The symbols have the same preferred meanings described above. 
     The lower alkyl groups represented by any of the variables include straight and branched chain hydrocarbon radicals from methyl to heptyl, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl and the like. The lower alkylene groups are of the same kind also having 1 to 7 carbons. Similarly, the lower alkoxy groups are of the same kind with a link to oxygen, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy and the like. The C 1  -C 4  members, especially C 1  and C 2  members, of all types are preferred. Phenylmethyl is the preferred phenyl-lower alkylene group and methoxy and t-butoxy the preferred lower alkoxy groups. The lower alkanoyl groups are the acyl radicals of the lower (up to 7 carbons) fatty acids, e.g., acetyl, propionyl, butyryl and the like, acetyl being preferred. 
     The amino(imino)methyl group represented by R 1  is the residue of the guanidino radical ##STR5## 
     The products of formula I and the preferred subgroups can be produced by various methods of synthesis. According to a preferred method, the amino acid of the formula ##STR6## wherein A, B and R are defined as above, and R is hydroxy is acylated with an acid of the formula ##STR7## wherein R 1  is an acyl group, and R 2 , R 3 , m and n have the meaning defined above, by one of the known procedures in which the acid IV is activated, prior to reaction with the amino acid III, involving formation of a mixed anhydride, symmetrical anhydride, acid chloride, active ester, Woodward reagent K, N,N&#39;-carbonylbisimidazole, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or the like. When R is lower alkoxy, this or other known methods of coupling such moieties can be used [For a review of these methods, see Methoden der Organischen Chemie (Houben-Weyl) Vol. XV, parts 1 and 2 (1974)]. 
     When the product obtained is an ester, e.g., R is t-butoxy, the ester can be converted to the free carboxy group (R is hydroxy) by cleavage with acids, e.g., trifluoroacetic acid. Conversely the free acid can be esterified by conventional procedures. 
     Starting materials of formula IV wherein m is 0 are derivatives of the amino acid cysteine which can be produced by known procedures. 
     The acids of formula IV wherein m is 2, 3 or 4 can be synthesized, according to a preferred method, by the addition of a thiol acid to the substituted acrylic acid of the formula ##STR8## 
     The latter are obtained by hydrolysis, e.g., with 6N hydrochloric acid, of the methylene lactams of the formula ##STR9## [J. Org. Chem. 39, 893 (1974)] 
     The compounds of formula I wherein m is 1 are obtained by the Curtius rearrangement of an acid of the formula ##STR10## 
     The preferred method for producing compounds of formula I wherein n is 0 is by displacement of the halo derivative of the formula ##STR11## wherein X is halogen, preferably chlorine or bromine, with the thiol acid R 3  --COSH. 
     The disulfides of formula I, wherein R 3  is ##STR12## are obtained by oxidation of the compound of the formula ##STR13## e.g., with an alcoholic solution of iodine. 
     The compounds of formula I wherein R 1  is amino(imino)-methyl are obtained by reacting a compound of formula I wherein R 1  or R 2  is hydrogen with a guanyl-forming reagent e.g., guanyl-3,5-dimethylpyrazole nitrate, S-methylisothiourea, or O-methylisourea. 
     Products of formula I have two asymmetric carbon atoms. These carbon atoms are indicated by an asterisk in formula I. The compounds accordingly exist in diastereoisomeric forms or in racemic mixtures thereof. All of these are within the scope of the invention. The above described syntheses can utilize the racemate or one of the enantiomers as starting material. When the racemic starting material is used in the synthetic procedure, the stereoisomers obtained in the product can be separated by conventional chromatographic or fractional crystallization methods. In general, the L-isomer with respect to the carbon of the amino acid constitutes the preferred isomeric form. 
     The compounds of formula I form salts which are also part of this invention. The salts include acid-addition salts which are formed by reaction with a variety of inorganic and organic acids providing acid addition salts including, for example, hydrohalides (especially hydrochloride and hydrobromide), sulfate, nitrate, borate, phosphate, oxalate, tartrate, maleate, citrate, acetate, ascorbate, succinate, benzenesulfonate, methanesulfonate, cyclohexanesulfamate and toluenesulfonate 
     The salts are formed in conventional manner by reacting the free form of the product with one or more equivalents of the appropriate acid or base providing the desired anion or cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freezing drying. By neutralizing the salt with an insoluble acid like a cation exchange resin in the hydrogen form [e.g., polystyrene sulfonic acid resin -- Dowex 50 (Mikes, Laboratory Handbook of Chromatographic Methods (Van Nostrand, 1961) page 256] eluting with a volatile buffer (e.g. pyridine-acetic acid) extraction with an organic solvent, the free form can be obtained, and if, desired, another salt formed. 
     Additional experimental details are found in the examples which are preferred embodiments and also serve as models for the preparation of other members of the group. 
     The compounds of this invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and therefore are useful in reducing or relieving angiotensin related hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance which has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g., rats, dogs, etc. The compounds of this invention intervene in the angiotensingen → angiotensin I → angiotensin II sequence by inhibiting angiotensin converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin II. 
     The inhibition of the angiotensin converting enzyme by compounds of formula I can be measured in vitro with isolated angiotensin converting enzyme from rabbit lungs following the procedure described by Cushman and Cheung [Biochem. Pharmacol., 20, 1637 (1971)], and with an excised smooth muscle assay [E. O&#39;Keefe, et al., Federation Proc. 31, 511 (1972)] in which these compounds have been shown to be powerful inhibitors of the contractile activity of angiotensin I and potentiators of the contractile activity of bradykinin. 
     The administration of a composition containing one or a combination of compounds of formula I or physiologically acceptable salt thereof to the species of hypertensive mammal alleviates or reduces hypertension. A single dose, or preferably two to four divided daily doses, provided on a basis of about 5 to 1000 mg. per kilogram per day, preferably about 10 to 500 mg. per kilogram per day is appropriate to reduce blood pressure. The animal model experiments described by S. L. Engel, T. R. SChaeffer, M. H. Waugh and B. Rubin, Proc. Soc. Exp. Biol. Med. 143, 483 (1973) serve as a useful guide. 
     The substance is preferably administered orally, but parenteral routes such as subcutaneously, intramuscularly, intravenously or intraperitoneally can also be employed. 
     The compounds of this invention can be utilized to achieve the reduction of blood pressure by formulating in compositions such as tablets, capsules or elixirs for oral administration or in sterile solution or suspensions for parenteral administration. About 10 to 500 mg. of a compound or mixture of compounds of formula I or physiologically acceptable salt is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.

The following examples are illustrative of the invention. Alltemperatures are in degrees celsius.

EXAMPLE 1N-tert-Butyloxycarbonyl-S-p-methoxybenzyl-D-cysteinyl-L-prolinetert-butyl ester.

To a solution of L-proline tert-butyl ester (0.85 g) andhydroxybenzotriazole (0.67 g) in methylene chloride (10 ml) chilled inan ice bath, dicyclohexylcarbodiimide (1.03 g) andN-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteine (1.7 g) are addedin that order. After fifteen minutes, the ice bath is removed and themixture is stirred at room temperature overnight. The precipitate isfiltered off and the filtrate is washed with 10% potassium bisulfate,water, saturated sodium bicarbonate, and water. The organic phase isdried and concentrated to dryness in vacuo to giveN-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteinyl-L-prolinetert-butyl ester as an oil. R_(f) =0.2 (silica gel, chloroform).

EXAMPLE 2 D-Cysteinyl-L-proline acetate

To a solution ofN-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteinyl-L-prolinetert-butyl ester (1.8 g) and anisole (4.4 ml) in dichloromethane (8 ml)chilled in an ice bath, trifluoromethane sulfonic acid (6.0 g) is added.The ice bath is removed and the mixture is stirred at room temperaturefor thirty minutes. The dichloromethane is removed in vacuo and theresidue is triturated with hexane (2 × 200 ml). The residue is dissolvedin water and extracted twice with ether. The aqueous phase is applied toa column of 200 ml of cation exchange resin [Doxex 50] in the hydrogencycle. The column is washed with water until no more acidic material iseluted. The D-cysteinyl-L-proline acetate is eluted with apyridine-acetic acid buffer pH 6.5, yield 0.66 g. R_(f) =0.38 (silicagel, chloroform:methanol:acetic acid:water).

EXAMPLE 3 N,S-Diacetyl-DL-cysteinyl-L-proline tert-butyl ester

By substituting N,S-diacetyl-DL-cysteine for theN-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteine in the procedure ofExample 1, N,S-diacetyl-DL-cysteinyl-L-proline tert-butyl ester isobtained. R_(f) =0.25 (silica gel, ethyl acetate).

EXAMPLE 4 N,S-Diacetyl-DL-cysteinyl-L-proline

N,S-Diacetyl-DL-cysteinyl-L-proline tert-butyl ester (1.9 g) isdissolved in a mixture of anisole (6 ml) and trifluoroacetic acid (12ml) and the solution is stored at room temperature for one hour. Thesolvents are removed in vacuo and the residue is precipitated from ethylacetate-ether-hexane, to obtain N,S-diacetyl-DL-cysteinyl-L-proline,yield 1.08 g, m.p. 80°-140°.

EXAMPLE 5 N-Acetyl-DL-cysteinyl-L-proline

N,S-Diacetyl-DL-cysteinyl-L-proline (0.3 g) is dissolved in a mixture ofwater (4 ml) and concentrated ammonia (4 ml) under a blanket of argon.The solution is stored for thirty minutes at room temperature, saturatedwith sodium chloride and extracted with ethyl acetate and chloroform.The organic layers are pooled and concentrated to dryness in vacuo toobtain N-acetyl-DL-cysteinyl-L-proline, yield 0.1 g, R_(f) =0.25 (silicagel; benzene:acetic acid, 75:25).

EXAMPLE 6 Methyl N-(p-methoxybenzyl)nipecotate hydrochloride

A mixture of 23 g of methyl nipecotate, 24.3 g of potassium carbonate,and 52 g of p-methoxybenzyl trichloroacetate in 800 ml of toluene isrefluxed under nitrogen for seventy-two hours. The mixture is cooled,the toluene removed in vacuo, the residue dissolved in chloroform, andthis solution washed once with 400 ml of aqueous potassium carbonate andthen with 400 ml of 10% hydrochloric acid. The chloroform solution isdried and concentrated in vacuo to a viscous brown oil. Trituration ofthis oil with ethyl acetate affords 30.7 g of methylN-(p-methoxybenzyl)nipecotate hydrochloride as an off-white crystallinesolid. Recrystallization from ethyl acetate yields the analyticalsample, m.p. 150°-154°.

EXAMPLE 7 1-(p-Methoxybenzyl)-3-methylene-2-piperidone

A solution of methyl N-(p-methoxybenzyl)nipecotate hydrochloride (30.7g) and 8.4 g of sodium hydroxide in 900 ml of methanol and 45 ml ofwater is stirred at room temperature for seventeen hours. The solutionis evaporated to dryness in vacuo, the residue diluted with toluene, andthis again evaporated to dryness in vacuo. To the residue is added 1liter of acetic anhydride and 140 ml of triethylamine, and the resultingmixture is heated under reflux for four hours. The reaction mixture isevaporated to dryness in vacuo, the residue taken up in chloroform,washed with water, dried, and concentrated in vacuo. The residual oil ischromatographed on silica gel using 1:1 hexane-ethyl acetate as theeluant, and yields 16.9 g of1-(p-methoxybenzyl)-3-methylene-2-piperidone as a chromatographicallypure yellow oil. Alternatively, the oil can be distilled to giveanalytically pure 1-(p-methoxybenzyl)-3-methylene-2-piperidone, b.p.145°-155°/0.05 mm.

EXAMPLE 8 3-Methylene-2-piperidone

A solution of 1-(p-methoxybenzyl)-3-methylene-2-piperidone (16.9 g) and21.3 g of anisole in 400 ml of trifluoroacetic acid is refluxed undernitrogen for forty-eight hours. The solution is evaporated to dryness invacuo, and the residue chromatographed on 900 g of silica gel usingethyl acetate as eluant, yielding 6.5 g of 3-methylene-2-piperidone as acrystalline solid.

EXAMPLE 9 2-Methylene-5-aminopentanoic acid hydrochloride

A solution of 2.6 g of 3-methylene-2-piperidone in 150 ml of 6Nhydrochloric acid is refluxed for 24 hours. The cooled solution isextracted with chloroform, and the aqueous layer concentrated in vacuoto 3.8 g of glassy foam. The foam is heated with methanol, filteredthrough Celite (diatomaceous earth clarifying agent) to remove a smallamount of insoluble material, and the filtrate is evaporated to drynessin vacuo, yielding 2.5 g of 2-methylene-5-aminopentanoic acidhydrochloride as a tan crystalline solid. Recrystallization fromisopropanol gives the analytical sample, m.p. 138°-144°.

EXAMPLE 10 2-Methylene-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid

To a solution of 8.8 g of 2-methylene-5-aminopentanoic acidhydrochloride in 100 ml of water is added with stirring 6.36 g ofmagnesium oxide, followed by a solution of 12.2 g ofp-methoxybenzyloxycarbonyl azide in 100 ml of dioxane, and the resultingmixture is stirred at room temperature for two days. The reactionmixture is filtered, and the filtrate diluted with 200 ml of ethylacetate, two equivalents of Dowex 50 ion exchange resin is added, andthe mixture is stirred at room temperature for two hours. The resin isthen filtered off and washed with water. The layers in the filtrate areseparated and the aqueous layer is extracted twice with ethyl acetate.The combined organic layers are dried and concentrated in vacuo to give18.2 g of 2-methylene-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acidas an amber oil which crystallizes on standing. This is used withoutfurther purification.

EXAMPLE 112-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid

A solution of 2-methylene-5-(p-methoxybenzyloxycarbonyl)amino pentanoicacid (53 mmoles) in 50 ml of thiolacetic acid is allowed to stand atroom temperature for forty-eight hours. The solution is evaporated todryness in vacuo, and the residue taken up in chloroform and applied toa silica gel column (700 g). Elution with 5% methanol in chloroformaffords 14.2 g of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid asan oil. Treatment of this oil with one equivalent of dicyclohexylaminein ether, followed by recrystallization from ethyl acetate affords thecorresponding dicyclohexylamine salt, m.p. 112°-114°.

EXAMPLE 12 2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid N-hydroxysuccinimide ester

To a solution of 3.7 g of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)aminopentanoic acid and1.21 g of N-hydroxysuccinimide in 60 ml of dichloromethane at 0°-5° isadded 2.16 g of N,N'-dicyclohexylcarbodiimide over 20 minutes withstirring. The resulting mixture is stirred overnight at 0°-5°. Theprecipitated dicyclohexylurea is filtered off, the filtrate concentratedin vacuo and the residue taken up in ethyl acetate and washed through asilica gel column to give 4.6 g of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonyl)amino pentanoic acidN-hydroxysuccinimide ester as an oil, which crystallizes on triturationwith ether. Recrystallization from ethyl acetate-hexane affords theanalytical sample, m.p. 85°-87°.

EXAMPLE 131-[(2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl)]-L-prolinetert-butyl ester

By substituting2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoic acid forthe N-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteine in theprocedure of Example 1,1-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolinetert-butyl ester is obtained.

EXAMPLE 14 1-(2-Acetylthiomethyl-5-aminopentanoyl)-L-prolinetrifluoroacetate

1-[2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolinetert-butyl ester (2 g) is dissolved in a mixture of trifluoroacetic acid(15 ml) and anisole (6 ml). The solution is stored at room temperaturefor one hour, the solvents are removed in vacuo and the residue isprecipitated from ethyl acetate-ether to yield1-(2-acetylthiomethyl-5-aminopentanoyl)-L-proline trifluoroacetate.

EXAMPLE 15 1-(5-Amino-2-mercaptomethylpentanoyl)-L-proline

1-(2-Acetylthiomethyl-5-aminopentanoyl)-L-proline trifluoroacetate (1 g)is dissolved in a mixture of water (12 ml) and concentrated ammonia (12ml) under a blanket of argon. The solution is stored twenty minutes atroom temperature concentrated to 5 ml and applied to a column of Dowex50 ion exchange resin in the hydrogen cycle. The column is washed withwater and 1-(5-amino-2-mercaptomethylpentanoyl)-L-proline is eluted witha buffer of pyridine-acetic acid at pH 6.5.

EXAMPLE 16 2-Methylene-4-methylaminobutanoic acid hydrochloride

By substituting 1-methyl-3-methylene-2-pyrrolidinone [J. Org. Chem., 39,893 (1974)] for the 3-methylene-2- piperidone in the procedure ofExample 9, 2-methylene-4-methylaminobutanoic acid hydrochloride isobtained.

EXAMPLE 172-Methylene-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)-butanoic acid

By substituting 2-methylene-4-methylaminobutanoic acid hydrochloride forthe 2-methylene-5-aminopentanoic acid hydrochloride in the procedure ofExample 10,2-methylene-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)-butanoic acidis obtained.

EXAMPLE 182-Acetylthiomethyl-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)butanoicacid

By substituting2-methylene-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)butanoic acidfor the 2-methylene-5-(p-methoxybenzyloxycarbonylamino)pentanoic acid inthe procedure of Example 11,2-acetylthiomethyl-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)butanoicacid is obtained.

EXAMPLE 19 1-(4-Amino-2-mercaptomethylbutanoyl)-L-proline

By substituting2-acetylthiomethyl-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)butanoicacid for the N-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteine in theprocedure of Example 1, and then submitting the product to the procedureof Examples 14 and 15,1-(2-acetylthiomethyl-4-(N-p-methoxybenzyloxycarbonyl-N-methylamino)butanoyl)-L-prolinetert-butyl ester, 1-(2-acetylthiomethyl-4-aminobutanoyl)-L-prolinetrifluoroacetate, and 1-(4-amino-2-mercaptomethylbutanoyl)-L-proline areobtained.

EXAMPLE 20 2-Acetylthiomethyl-6-(N-methyl-N-acetylamino)hexanoic acid

By substituting 6-(N-methyl-N-acetylamino)-2-methylene hexanoic acid forthe 2-methylene-5-(p-methoxybenzyloxycarbonylamino)pentanoic acid in theprocedure of Example 11,2-acetylthiomethyl-6-(N-methyl-N-acetylamino)hexanoic acid is obtained.

EXAMPLE 211-[(2-Mercaptomethyl-6-(N-methyl-N-acetylamino)hexanoyl]-L-proline

By substituting 2-acetylthiomethyl-6-(N-methyl-N-acetylamino)hexanoicacid for the N-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteine in theprocedure of Example 1, and then submitting the product to the procedureof Examples 14 and 15,1-[2-acetylthiomethyl-6-(N-methyl-N-acetylamino)-hexanoyl]-L-prolinetert-butyl ester,1-[2-acetylthiomethyl-6-(N-methyl-N-acetylamino)hexanoyl]-L-proline, and1-[2-mercaptomethyl-6-(N-methyl-N-acetylamino)hexanoyl]-L-proline areobtained.

EXAMPLE 22 1-(5-Guanidino-2-mercaptomethylpentanoyl)-L-proline

A solution of 1-(5-amino-2-mercaptomethylpentanoyl)-L-proline (2.3 g),guanyl-3,5-dimethylpyrazole nitrate (2.41 g) and triethylamine (3.36 ml)in dimethylformamide (20 ml) is stored at room temperature under ablanket of argon for 16 hours. The solvents are removed in vacuo, theresidue is dissolved in 0.1 N hydrochloric acid (10 ml) and zinc dust(500 mg) is added. The suspension is stirred at room temperature for twohours. After filtering, the filtrate is applied to a column of Dowex 50ion exchange resin in the hydrogen cycle. The column is washed withwater until no more acid is eluted and1-(5-guanidino-2-mercaptomethylpentanoyl)-L-proline is then eluted witha pyridine-acetate buffer at pH 6.5.

EXAMPLE 23 N-(5-Amino-2-mercaptomethylpentanoyl)glycine

By substituting glycine tert-butyl ester for the proline tert-butylester in the procedure of Example 13, and then submitting the product tothe procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]glycinetert-butyl ester, N-[2-acetylthiomethyl-5-aminopentanoyl]glycine andN-(5-amino-2-mercaptomethylpentanoyl)glycine are obtained.

EXAMPLE 24 N-(5-Amino-2-mercaptomethylpentanoyl)-L-leucine

By substituting L-leucine tert-butyl ester for the proline tert-butylester in the procedure of Example 13, and then submitting the product tothe procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-leucinetert-butyl ester, N-[2-acetylthiomethyl-5-aminopentanoyl]-L-leucine andN-(5-amino-2-mercaptomethylpentanoyl)-L-leucine are obtained.

EXAMPLE 25 N-(5-Amino-2-mercaptomethylpentanoyl)-L-phenylalanine

By substituting L-phenylalanine tert-butyl ester for the L-prolinetert-butyl ester in the procedure of Example 13, and then submitting theproduct to the procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-phenylalaninetert-butyl ester,N-[2-acetylthiomethyl-5-aminopentanoyl)-L-phenylalanine andN-(5-amino-2-mercaptomethylpentanoyl)-L-phenylalanine are obtained.

EXAMPLE 26 N-(5-Amino-2-mercaptomethylpentanoyl)-L-serine

By substituting O-tert-butyl L-serine tert-butyl ester for the L-prolinetert-butyl ester in the procedure of Example 13, and then submitting theproduct to the procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-O-tert-butylL-serine tert-butyl ester,N-(2-acetylthiomethyl-5-aminopentanoyl)-L-serine andN-(5-amino-2-mercaptomethylpentanoyl)-L-serine are obtained.

EXAMPLE 27 1-(5-Amino-2-mercaptomethylpentanoyl)-4-hydroxy-L-proline

By substituting 4-hydroxy-L-proline p-methoxybenzyl ester for theL-proline tert-butyl ester in the procedure of Example 13, and thensubmitting the product to the procedures of Examples 14 and 15,1-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-4-hydroxy-L-prolinep-methoxybenzyl ester,1-(2-acetylthiomethyl-5-aminopentanoyl)-4-hydroxy-L-proline, and1-(5-amino-2-mercaptomethylpentanoyl)-4-hydroxy-L-proline are obtained.

EXAMPLE 28 1-(5-Amino-2-mercaptomethylpentanoyl)pipecolic acid

By substituting pipecolic acid tert-butyl ester (obtained from pipecolicacid by the procedure described for the synthesis of L-prolinetert-butyl ester) for the L-proline tert-butyl ester in the procedure ofExample 13 and then submitting the product to the procedure of Examples14 and 15,1-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]pipecolicacid tert-butyl ester, 1-(2-acetylthiomethyl-5-aminopentanoyl)pipecolicacid and 1-(5-amino-2-mercaptomethylpentanoyl)pipecolic acid areobtained.

EXAMPLE 29 N.sup.α-[2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)-pentanoyl]-L-arginine

A solution of2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoic acidN-hydroxysuccinimide ester (4.6 g) in ethanol (16 ml) is added to asolution of L-arginine (1.47 g) in a mixture of water (15 ml) and sodiumbicarbonate (1.68 g). The mixture is stirred at room temperature foreight hours, acidified to pH 3 and extracted with ethyl acetate. Theaqueous phase is applied to a column of Dowex 50 ion exchange resin (100ml) in the hydrogen cycle. The column is washed with water until no moreacidic material is eluted and then N.sup.α-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-arginineis eluted with pyridine-acetate buffer at pH 6.5.

EXAMPLE 30 N.sup.α -(2-Acetylthiomethyl-5-aminopentanoyl)-L-argininetrifluoroacetate

A solution of N.sup.α-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-arginine(1 g) in trifluoroacetic acid (10 ml) is stored at room temperature forfifteen minutes, and then concentrated to dryness in vacuo to yieldN.sup.α -(2-acetylthiomethyl-5-aminopentanoyl)-L-argininetrifluoroacetate.

EXAMPLE 31 N.sup.α -(5-Amino-2-mercaptomethylpentanoyl)-L-arginine

N.sup.α -(2-acetylthiomethyl-5-aminopentanoyl)-L-argininetrifluoroacetate (2 g) is dissolved in a mixture of water (25 ml) andconcentrated ammonia (25 ml) and this solution is stored at roomtemperature for twenty minutes. The solution is concentrated in vacuo toca. 5 ml and applied to a column of Dowex 50 ion exchange resin (50 ml)in the hydrogen cycle. After washing with water N.sup.α-(5-amino-2-mercaptomethylpentanoyl)-L-arginine is eluted with apyridine-acetate buffer at pH 6.5.

EXAMPLE 32 N.sup.α-[2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)-pentanoyl]-N.sup.ε-tert-butyloxycarbonyl-L-lysine tert-butyl ester

By substituting N.sup.ε -tert-butyloxycarbonyl lysine tert-butyl esterfor the L-proline tert-butyl ester in the procedure of Example 13,N.sup.α-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-N.sup..epsilon.-tert-butyloxycarbonyl-L-lysine tert-butyl ester is obtained.

EXAMPLE 33 N.sup.α -(2-Acetylthiomethyl-5-aminopentanoyl)-L-lysinetrifluoroacetate

A solution of N.sup.α-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-N.sup..epsilon.-tert-butyloxycarbonyl-L-lysine tert-butyl ester (1 g) intrifluoroacetic acid (5 ml) is stored at room temperature for one hourand then concentrated to dryness in vacuo to yield N.sup.α-(2-acetylthiomethyl-5-aminopentanoyl)-L-lysine trifluoroacetate.

EXAMPLE 34 N.sup.α -(5-Amino-2-mercaptomethylpentanoyl)-L-lysine

By substituting N.sup.α -(2-acetylthiomethyl-5-aminopentanoyl)-L-lysinetrifluoroacetate for the N.sup.α-(2-acetylthiomethyl-5-aminopentanoyl)-L-arginine in the procedure ofExample 31, N.sup.α -(5-amino-2-mercaptomethylpentanoyl)-L-lysine isobtained.

EXAMPLE 35 N.sup.α -(5-Amino-2-mercaptomethylpentanoyl)-L-histidine

By substituting L-histidine for the L-arginine in the procedure ofExample 29 and then submitting the product to the procedures of Examples30 and 31, N.sup.α-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-histidine,N.sup.α -(2-acetylthiomethyl-5-aminopentanoyl)-L-histidine, and N.sup.α-(5-amino-2-mercaptomethylpentanoyl)-L-histidine are obtained.

EXAMPLE 36 N-(5-Amino-2-mercaptomethylpentanoyl)-L-methionine

By substituting L-methionine diphenylmethyl ester for the L-prolinetert-butyl ester in the procedure of Example 13, and then submitting theproduct to the procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-methioninediphenylmethyl ester,N-(2-acetylthiomethyl-5-aminopentanoyl)-L-methionine, andN-(5-amino-2-mercaptomethylpentanoyl)-L-methionine are obtained.

EXAMPLE 37N-[2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)-pentanoyl]-L-tryptophanemethyl ester

A solution of L-tryptophane methyl ester hydrochloride (2.5 g),2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoic acidN-hydroxysuccinimide ester (4.6 g), and hydroxybenzotriazole (1.35 g) ina mixture of dimethylformamide (20 ml) and triethylamine (1.6 ml) isstored at room temperature overnight. The solvent is removed in vacuo,the residue is dissolved in ethyl acetate and washed neutral. Theorganic phase is dried and concentrated to dryness to yieldN-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-tryptophanemethyl ester.

EXAMPLE 38 N-(2-Acetylthiomethyl-5-aminopentanoyl)-L-tryptophane methylester trifluoroacetate

N-[2-Acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-tryptophanemethyl ester (1 g) is dissolved in trifluoroacetic acid (10 ml), thesolution is stored at room temperature for fifteen minutes, and thenevaporated in vacuo to yieldN-(2-acetylthiomethyl-5-aminopentanoyl)-L-tryptophane methyl estertrifluoroacetate.

EXAMPLE 39 N-(5-Amino-2-mercaptomethylpentanoyl)-L-tryptophane

To a solution of N-(2-acetylthiomethyl-5-aminopentanoyl)-L-tryptophanemethyl ester trifluoroacetate (3 g) in methanol (60 ml) N sodiumhydroxide (60 ml) is added. After four hours the solution is applied toa column of Dowex 50 ion exchange resin in the hydrogen cycle. Afterwashing with water, theN-(5-amino-2-mercaptomethylpentanoyl)-L-tryptophane is eluted withpyridine-acetic acid buffer at pH 6.5.

EXAMPLE 40 N-(5-Amino-2-mercaptomethylpentanoyl-L-glutamine

By substituting L-glutamine tert-butyl ester for the L-prolinetert-butyl ester in the procedure of Example 13, and then submitting theproduct to the procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-glutaminetert-butyl ester, N-(2-acetylthiomethyl-5-aminopentanoyl)-L-glutamine,and N-(5-amino-2-mercaptomethylpentanoyl-L-glutamine are obtained.

EXAMPLE 41 N-(5-Amino-2-mercaptomethylpentanoyl)-L-aspartic acid

By substituting L-aspartic acid di tert-butyl ester for the L-prolinetert-butyl ester in the procedure of Example 13, and then submitting theproduct to the procedures of Examples 14 and 15,N-[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanonyl]-L-asparticdi tert-butyl ester, N-(2-acetylthiomethyl-5-aminopentanonyl)-L-asparticacid, and N-(5-amino-2-mercaptomethylpentanoyl)-L-aspartic acid areobtained.

EXAMPLE 42 D-Cysteinyl-L-alanine

By substituting L-alanine tert-butyl ester for the L-proline tert-butylester in the procedure of Example 1, and then submitting the product tothe procedure of Example 2, D-cysteinyl-L-alanine is obtained.

EXAMPLE 43 D-Cysteinyl-L-phenylglycine

By substituting L-phenylglycine tert-butyl ester (prepared fromL-phenylglycine by the procedure described for L-proline tert-butylester) for the L-proline tert-butyl ester in the procedure of Example 1,and then submitting the product to the procedure of Example 2,D-cysteinyl-L-phenylglycine is obtained.

EXAMPLE 44 D-Cysteinyl-L-threonine

By substituting O-tert-butyl-L-threonine tert-butyl ester for theL-proline tert-butyl ester in the procedure of Example 1 and thensubmitting the product to the procedure of Example 2,D-cysteinyl-L-threonine is obtained.

EXAMPLE 45

N-(5-Guanidino-2-mercaptomethylpentanoyl)-L-phenylalanine

By substituting N-(5-amino-2-mercaptomethylpentanoyl)-L-phenylalaninefor the 1-(5-amino-2-mercaptomethylpentanoyl)-L-proline in the procedureof Example 22, N-(5-guanidino-2-mercaptomethylpentanoyl)-L-phenylalanineis obtained.

EXAMPLE 46 N-(5-Guanidino-2-mercaptomethylpentanoyl)-L-leucine

By substituting N-(5-amino-2-mercaptomethylpentanoyl-L-leucine for the1-(5-amino-2-mercaptomethylpentanoyl)-L-proline in the procedure ofExample 22, N-(5-guanidino-2-mercaptomethylpentanoyl)-L-leucine isobtained.

EXAMPLE 47 3-Acetylthio-2-methoxycarbonylmethylpropanoic acid

A mixture of thiolacetic acid (12.5 g) and3-methoxycarbonyl-2-methylenepropanoic acid (17.1 g) are heated on thesteam bath for two hours. The reaction is concentrated in vacuo and theresidue is dissolved in ethyl acetate (125 ml) and dicyclohexylamine (35ml) is added. The crystals are filtered, dried and recrystallized fromethyl acetate to yield 37.8 g, of the dicyclohexylammonium salt of3-acetylthio-2-methoxycarbonylmethylpropanoic acid, m.p. 120°-121°. Thisdicyclohexylammonium salt is converted to the free acid,3-acetylthio-2-methoxycarbonylmethylpropanoic acid, by distributionbetween ethyl acetate and 10% aqueous potassium bisulfate.

EXAMPLE 48 1-[3-(Acetylthio)-2-methoxycarbonylmethylpropanoyl]-L-prolinetert-butyl ester

To a solution of L-proline tert-butyl ester (1.71 g) and3-hydroxybenzotriazole (1.35 g) in dichloromethane (15 ml),dicyclohexylcarbodiimide (2.06 g) and3-acetylthio-2-methoxycarbonylmethylpropanoic acid (2.2 g) are added.After eighteen hours stirring at room temperature, the precipitateformed is filtered off, the filtrate is washed neutral, dried, andconcentrated to dryness to yield 3.7 g of1-[3-(acetylthio)-2-methoxycarbonylmethylpropanoyl]-L-proline tert-butylester. R_(f) =0.8 (silica gel-ethyl acetate).

EXAMPLE 49 1-[3-(Acetylthio)-2-carboxymethylpropanoyl]-L-prolinetert-butyl ester

To a solution of1-[3-(acetylthio)-2-methoxycarbonylmethylpropanoyl]-L-proline tert-butylester (3.7 g) in methanol (60 ml), N sodium hydroxide (40 ml) is added.After four hours, the reaction mixture is diluted with water (100 ml)and extracted with ethyl acetate. The aqueous layer is acidified andextracted with ethyl acetate. This last ethyl acetate layer is dried andconcentrated to dryness in vacuo. The residue is dissolved in a mixtureof pyridine and acetic anhydride (3:1) and the solution is stored atroom temperature overnight. The reaction mixture is diluted with ethylacetate (200 ml) and washed with 10% potassium bisulfate. The organiclayer is dried and concentrated to dryness in vacuo to yield1-[3-(acetylthio)-2-carboxymethylpropanoyl]-L-proline tert-butyl ester.

EXAMPLE 501-[3-(Acetylthio)-2-tert-butyloxycarbonylaminomethylpropanoyl]-L-prolinetert-butyl ester

To a solution of 1-[3-(acetylthio)-2-carboxymethylpropanoyl]-L-prolinetert-butyl ester (3.6 g) in tert-butanol (60 ml), triethylamine (1.4 ml)and diphenylphosphoryl azide (2.75 g) are added. The mixture is refluxedfor twenty-two hours, and concentrated to dryness in vacuo. The residueis dissolved in ethyl acetate and the solution is washed neutral. Theorganic phase is dried and concentrated to dryness in vacuo to yield1-[3-(acetylthio)-2-tert-butyloxycarbonylaminomethylpropanoyl]-L-prolinetert-butyl ester.

EXAMPLE 51 1-(3-Acetylthio-2-aminomethylpropanoyl)-L-proline

1-[3-(acetylthio)-2-tert-butyloxycarbonylaminomethylpropanoyl]-L-prolinetert-butyl ester (1.5 g) is dissolved in a mixture of anisole (6 ml) andtrifluoroacetic acid (12 ml) and the solution is stored at roomtemperature for one hour. The solvent is removed in vacuo, the residueis distributed between water and ether. The aqueous phase is washedtwice with ether and freeze-dried to yield1-(3-acetylthio-2-aminomethylpropanoyl)-L-proline.

EXAMPLE 52 -(2-Aminomethyl-3-mercaptopropanoyl)-L-proline

By substituting 1-(3-acetylthio-2-aminomethylpropanoyl)-L-proline forthe 1-(2-acetylthiomethyl-5-aminopentanoyl)-L-proline trifluoroacetatein the procedure of Example 15,1-(2-aminomethyl-3-mercaptopropanoyl)-L-proline is obtained.

EXAMPLE 53 1-(5-Guanidino-2-mercaptomethylpentanoyl)pipecolic acid

By substituting 1-(5-amino-2-mercaptomethylpentanoyl)-pipecolic acid forthe 1-(5-amino-2-mercaptomethylpentanoyl)-L-proline in the procedure ofExample 22, 1-(5-guanidino-2-mercaptomethylpentanoyl)pipecolic acid isobtained.

EXAMPLE 54 1-(5-Guanidino-2-mercaptomethylpentanoyl)-4-hydroxy-L-proline

By substituting1-(5-amino-2-mercaptomethylpentanoyl)-4-hydroxy-L-proline for the1-(5-amino-2-mercaptomethyl-pentanonyl)-L-proline in the procedure ofExample 22,1-(5-guanidino-2-mercaptomethylpentanoyl)-4-hydroxy-L-proline isobtained.

EXAMPLE 55 1,1'-Dithiobis-(2-D-amino-3-propanoyl)-bis-L-proline

An alcoholic solution of iodine is added dropwise to a solution ofD-cysteine-L-proline (1 g.) in water (10 ml.) while keeping the pHbetween 5 and 7 by careful addition of N sodium hydroxide. When apersistent yellow color is obtained, the solution is applied to a columnof Dowex 50 ion exchange resin in the hydrogen cycle and washed withwater. The 1,1'-dithiobis-(2-D-amino-3-propanoyl)-bis-L-proline iseluted with a pyridine-buffer at pH 6.5.

EXAMPLE 561-[2-Mercaptomethyl-5-(p-methoxybenzyloxycarbonylamino)-pentanoyl]-L-proline

By substituting1[2-acetylthiomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolinefor the N,S-diacetyl-DL-cysteinyl-L-proline in the procedure of Example5,1-[2-mercaptomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolineis obtained.

EXAMPLE 571,1'-Dithiobis-[2-(p-methoxybenzyloxycarbonylaminopropyl)-3-propanoyl]-bis-L-proline

By substituting1-[2-mercaptomethyl-5-(p-methoxybenzyloxycarbonylamino)pentanoyl]-L-prolinefor the D-cysteinyl-L-proline in the procedure of Example 55,1,1'-dithiobis-[2-(p-methoxybenzyloxycarbonylaminopropyl)-3-propanoyl]-bis-L-prolineis obtained.

EXAMPLE 58 1,1'-Dithiobis-(2-aminopropyl-3-propanoyl)-bis-L-proline

By substituting1,1'-dithiobis-[2-(p-methoxybenzyloxycarbonylaminopropyl)-3-propanoyl]-bis-L-prolinefor the N.sup.α-[2-acetylthiomethyl-5-(p-methoxybenzylcarbonylamino)-pentanoyl]-L-argininein the procedure of Example 30,1,1'-dithiobis-(2-aminopropyl-3-propanoyl)-bis-L-proline is obtained.

EXAMPLE 59 1,1'-Dithiobis-(2-aminopropyl-3-propanoyl)-bis-L-leucine

By substituting N-(5-amino-2-mercaptomethylpentanoyl)-L-leucine for theD-cysteine-L-proline in the procedure of Example 55,1,1'-dithiobis-(2-aminopropyl-3-propanoyl)-bis-L-leucine is obtained.

EXAMPLE 60 L-Cysteinyl-L-proline acetate

By substituting N-tert-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteine forthe N-tert-butyloxycarbonyl-S-p-methoxybenzyl-D-cysteine in theprocedure of Example 1 and submitting the product to the procedure ofExample 2,N-tert-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-L-prolinetert-butyl ester and L-cysteinyl-L-proline acetate are obtained.

What is claimed is:
 1. A compound of the formula ##STR14## and salts thereof, wherein R is hydroxy or lower alkoxy; R₁ is hydrogen, lower alkanoyl or amino(imino)methyl;R₂ is hydrogen, lower alkyl or phenyl-lower alkylene; and R₃ is hydrogen, lower alkanoyl, benzoyl or ##STR15##
 2. A compound as claimed in claim 1 wherein R is hydroxy.
 3. A compound as in claim 1 wherein R is hydroxy and R₁, R₂ and R₃ each is hydrogen.
 4. A compound as in claim 1 wherein R is hydroxy; R₁ is hydrogen, lower alkanoyl or amino(imino)methyl; R₂ is hydrogen, lower alkyl or phenyl-lower alkylene; and R₃ is hydrogen, lower alkanoyl or benzoyl.
 5. A compound as in claim 1 wherein R₃ is ##STR16##
 6. A compound as in claim 5 wherein each R is hydroxy and each R₁ and R₂ is hydrogen.
 7. Cysteinylproline and acid addition salts thereof.
 8. Acetate salt of compound of claim
 7. 9. D-cysteinyl-L-proline. 